Cholinergic neurons in the medial septum project their axons to the hippocampus, and are critically involved in producing stable network oscillations underlying many cognitive functions. Although the distribution and pharmacological profiles of nicotinic and muscarinic acetylcholine (ACh) receptors (nAChRs and mAChRs, respectively) in the hippocampus are well-known, less is understood about the regulation of cholinergic transmission and the actions of the neurotransmitter ACh. Endogenous cannabinoids (endocannabinoids) are retrograde signaling molecules in the central nervous system. They are released from a post-synaptic cell via several mechanisms, including activation of mAChRs, and suppress transmitter release via activation of presynaptic cannabinoid-1 receptors (CB1Rs). Prior evidence shows that GABAergic interneurons in the hippocampus that co-express the neuropeptide cholecystokinin (CCK+ interneurons), also highly express cholinergic receptors, can release endocannabinoids, and may be intermediaries through which septal cholinergic neurons drive hippocampal theta-frequency oscillations. This proposal will test the broad hypothesis that cholinergic modulation of hippocampal networks is regulated by endocannabinoids. To test key predictions of this hypothesis, Channelrhodopsin-2, a light-activated cation channel, will be expressed in septal cholinergic cells and used to control the release of ACh from axons in mouse hippocampal slices. By combining patch-clamp electrophysiology with pharmacological tools already established for studying endocannabinoid signaling, it will be determined if endocannabinoids regulate ACh release onto CCK+ interneurons (Specific Aim 1). Specific Aim 2 will determine the role of endocannabinoid signaling in the control of ACh-induced activation of CCK+ interneurons, and Specific Aim 3 will investigate how endocannabinoids regulate cholinergically-driven theta frequency inhibition in hippocampal CA1 pyramidal cells. The results will establish a new optogenetic model for the study of endogenous ACh in hippocampal slice preparations, contribute to our understanding of the cellular mechanisms of learning and memory, and provide insight into the consequences of cholinergic dysfunction in several neurological diseases, such as Alzheimer's, epilepsy, and autism. PUBLIC HEALTH RELEVANCE: In the mammalian brain, the hippocampus is critically important for learning and memory. This proposal will investigate the interaction between two signaling molecules in the hippocampus, acetylcholine and endogenous cannabinoids, both of which may contribute to the dysfunction of learning and memory in common neurological diseases such as Alzheimer's.